Summary Shortage of available allogeneic donor organs represents the greatest unmet medical need in the field of transplantation today. The survival of pig-to-primate organ transplants has improved markedly over the past three decades but unfortunately remains insufficient for clinical application. Further progress in this field will likely depend upon genetic modifications of porcine source animals both to overcome molecular incompatibilities with primate recipients and to allow the induction of immunological tolerance. The objectives of this proposal are to produce such pigs and to identify additional genetic modifications that will help attain the goal of clinically relevant xenograft survival. Advanced genetic engineering techniques, including site-specific recombinase-driven gene additions and edits using the precision CRISPR-Cas9 system, will be used to effectively deliver multiple genetic modifications affecting complement activation, coagulation and barriers to induction of mixed hematopoietic chimerism to achieve tolerance. These modifications will be made efficiently and rapidly by applying state-of-the-art genomic sequencing and guide RNA design specifically tailored to the highly inbred MGH miniature swine to be engineered. Genetically modified pigs incorporating the new modifications will be tested in Projects 1,2, and 3 for efficacy in promoting organ survival and mixed chimerism for tolerance induction in primate and humanized mouse models. Additional advanced technology will be used to generate millions of random pig knockout/human transgenic genotypes and to identify those that enhance survival of a porcine hematopoietic cell line in primates. This unbiased discovery effort will provide the foundation for the creation of additional transgenic animals that will better meet the needs for eventual clinical use as organ, tissue and cell donors.